Inducible expression of filaggrin increases keratinocyte susceptibility to apoptotic cell death

Filaggrin is an intermediate filament associated protein that aids the packing of keratin filaments during terminal differentiation of keratinocytes. Premature aggregation of keratin filaments is prevented by filaggrin expression as the inactive precursor, profilaggrin, which is localized in keratohyalin granules in vivo. We have previously shown that filaggrin constructs, when transiently transfected into epithelial cells, lead to a collapsed keratin cytoskeletal network and dysmorphic nuclei with features of apoptosis. The apparent transfection rate is low with filaggrin constructs, supporting their disruptive role but hindering further study. To bypass this problem, we generated stable keratinocyte cell lines that express mature human filaggrin using a tetracycline-inducible promoter system. We found that cell lines expressing filaggrin, but not control cell lines, exhibited increased sensitivity to multiple apoptotic stimuli as measured by morphologic and biochemical criteria. None of the cell lines showed an increase in endogenous expression of filaggrin in response to the same stimuli. Filaggrin expression alone was insufficient to induce apoptosis in these keratinocyte cell lines. We conclude that filaggrin, due to its keratin binding ability, primes cells for apoptosis. Because filaggrin is expressed at a level of the epidermis where keratinocytes are in transition between the nucleated granular and the anucleate cornified layers, we hypothesize that filaggrin aids in the terminal differentiation process by facilitating apoptotic machinery.     

Lipid constituents in oligodendroglial cells alter susceptibility to H2O2-induced apoptotic cell death via ERK activation

The present work examines the effect of membrane lipid composition on activation of extracellular signal-regulated protein kinases (ERK) and cell death following oxidative stress. When subjected to 50 microM docosahexaenoic acid (DHA, 22 : 6 n-3), cellular phospholipids of OLN 93 cells, a clonal line of oligodendroglia origin low in DHA, were enriched with this polyunsaturated fatty acid. In the presence of 1 mM N,N-dimethylethanolamine (dEa) a new phospholipid species analog was formed in lieu of phosphatidylcholine. Exposure of DHA-enriched cells to 0.5 mM H2O2, caused sustained activation of ERK up to 24 h. At this time massive apoptotic cell death was demonstrated by ladder and TUNEL techniques. H2O2-induced stress applied to dEa or DHA/dEa co-supplemented cells showed only a transient ERK activation and no cell death after 24 h. Moreover, while ERK was rapidly translocated into the nucleus in DHA-enriched cells, dEa supplements completely blocked ERK nuclear translocation. This study suggests that H2O2-induced apoptotic cell death is associated with prolonged ERK activation and nuclear translocation in DHA-enriched OLN 93 cells, while both phenomena are prevented by dEa supplements. Thus, the membrane lipid composition ultimately modulates ERK activation and translocation and therefore can promote or prevent apoptotic cell death.     

Characterization of ceramide-induced apoptotic death in cerebellar granule cells in culture

Sphingomyelin signalling system has been involved in several examples of cell death through apoptosis. We have characterised the effect of exposure to the cell permeable ceramide analogue, C2-ceramide, on cultures of differentiated cerebellar granule cells. C(2)-ceramide was toxic to granule cells in a dose- and time-dependent way at concentrations higher than 10 microM. Ceramide exposure was accompanied by characteristic alterations of cell morphology, namely swollen cell bodies and punctuate appearance and arcuate direction of processes. The final outcome of ceramide exposure was a form of cell death largely apoptotic in nature. Hoechst stain, followed by counts of nuclei with normal appearance and size or with condensed chromatin and reduced size, revealed a large increase of the proportion of shrunken nuclei in treated cultures. In situ visualisation of fragmented DNA through the TUNEL technique, additionally marked cells undergoing apoptosis as a consequence of ceramide treatment. Accordingly, the DNA extracted from cultures exposed to C2-ceramide and subjected to agarose gel electrophoresis showed the peculiar ladder of fragmented low molecular weight DNA. Treatments with inhibitors of two caspases or of nitric oxide synthase were unable to rescue neurons exposed to ceramide, thus suggesting a neurotoxic action not primarily dependent on activation of death proteases or on nitric oxide production.     

Downregulation of Bcr-Abl in K562 cells restores susceptibility to apoptosis: characterization of the apoptotic death

We examined the susceptibility of a variety of human leukemic cell lines to the induction of apoptosis. K562, a chronic myelogenous leukemic cell line which expresses the bcr-abl fusion gene, was found to be extremely resistant to apoptosis, irrespective of the inducing agent. This resistance can be attributed to the deregulated Abl kinase activity of bcr-abl, as downregulation of its expression using antisense oligodeoxynucleotides targeted to the beginning of the abl sequence in this chimeric gene rendered these cells susceptible to cytotoxic drug-induced apoptosis. Examination of the morphological and biochemical features of apoptosis in K562 cells revealed the typical membrane blebbing and chromatin condensation associated with this form of cell death. In situ TdT-mediated end labeling of the DNA revealed the presence of strand breaks in the treated cells and field inversion gel electrophoresis revealed the presence of large 10-50 kb fragments. However there was an absence of oligonucleosomal DNA fragmentation, whether or not Bcr-Abl was expressed. Thus, while inhibition of expression of Bcr-Abl renders K562 cells susceptible to apoptosis, the absence of oligonucleosomal DNA fragmentation in these cells is independent of the function of this molecule.     

Kolaviron protects apoptotic cell death in PC12 cells exposed to atrazine

Kolaviron (KV), a natural biflavonoid obtained from the seeds of Garcinia kola, has been documented for its wide pharmacological window, including anti-apoptotic activities. However, the underlying mechanisms are poorly understood at the cellular level. This study investigates the anti-apoptotic activity of KV in PC12 cells, a rat pheochromocytoma, exposed to endocrine disruptor-atrazine (ATZ). KV (60 μM) treatment for 24 h shows significant anti-apoptotic responses in PC12 cells exposed to ATZ (232 μM) for 24 h. KV treatment recovers the ATZ-induced levels of malondialdehyde, reactive oxygen species (ROS), caspase-3 activity and depleted levels of glutathione and catalase activity. However, KV was found to be ineffective to restore the ATZ-induced expression (mRNA) and activity of glutathione-peroxidase (GSH-Px) and glutathione reductase (GR). KV treatment also demonstrates significant restoration in ATZ-induced alterations in the expression of apoptosis markers viz., p53, Bax, Bcl2, caspase-3, caspase-9, cyclooxygenase-2 (COX-2), c-Jun and c-fos. Flow cytometric analysis confirms the involvement of ROS in the mediation of ATZ-induced apoptosis in PC12 cells. Together, these data suggest that KV has the therapeutic potential against chemical-induced apoptotic cell death in the neuronal system.     

Mechanisms of photochemotherapy-induced apoptotic cell death in lymphoid cells.

Previous studies we performed showed that 8-methoxypsoralen in combination with ultraviolet A light (photochemotherapy) caused DNA damage and that this caused nucleotide depletion in peripheral blood leukocytes, secondary to an active form of programmed cell death, poly(ADP-ribosyl)ation. Further studies revealed that 24 h after exposure to 10 J/cm2 ultraviolet A light and 8-methoxypsoralen (300 ng/mL), apoptotic cells increased from 3 (control) to 31% (p less than 0.001). Ultraviolet A light alone also significantly increased the number of apoptotic cells. These morphological changes were confirmed by parallel findings on DNA electrophoresis. Treatment with 2 to 5 J/cm2 of ultraviolet A light and 8-methoxypsoralen caused an approximately 30% increase in cytosolic free calcium levels in peripheral blood leukocytes 1 h after exposure. Associated with this was a 51% increase in 45Ca2+ uptake over the first 60 min. Similar findings in a different lymphoid cell (CCRF-CEM) confirmed the results obtained with peripheral blood leukocytes. The use of calcium-free medium prevented a rise in cytosolic free calcium and decreased the number of cells undergoing apoptotic cell death. Cycloheximide inhibited ultraviolet A light - 8-methoxypsoralen induced apoptosis in CCRF-CEM cells; it also decreased calcium levels in control CCRF-CEM cells. This study shows that ultraviolet A light - 8-methoxypsoralen caused apoptotic cell death in lymphoid cells; this appeared to be associated with calcium influx, presumably because of the requirement of endogenous endonucleases for calcium.     

Intracellular free calcium related to apoptotic cell death in quail granulosa cell sheets kept in serum-free culture

The relationship between apoptosis and resting intracellular free calcium ([Ca2+]i) was studied in serum-free cultures of granulosa cell sheets isolated from preovulatory quail follicles. Apoptosis was detected by acridine orange, in situ end-labeling of fragmented DNA and electron microscopy. [Ca2+]i was measured using fura-2. [Ca2+]i averaged 525 mM in freshly isolated sheets. In 24 h cultures no apoptosis was detected but [Ca2+]i became very dispersed, 20% of the sheets showing values above 1000 nM. At 48 h, apoptosis was obvious and [Ca2+]i remained dispersed. At 72 h, apoptosis and also the fraction of sheets with high [Ca2+]i were at their maximum. At 96 h apoptosis was subsiding and [Ca2+]i normalized. FSH depressed apoptosis and [Ca2+]i in the 72 h cultures. We conclude that at 24 h apoptosis is initiated at high [Ca2+]i foci. At later stages apoptosis is associated with high [Ca2+]i, but it is not clear whether this is cause or consequence.     

Mitochondrial regulation of apoptotic cell death

Mitochondria play a decisive role in the regulation of both apoptotic and necrotic cell death. Permeabilization of the outer mitochondrial membrane and subsequent release of intermembrane space proteins are important features of both models of cell death. The mechanisms by which these proteins are released depend presumably on cell type and the nature of stimuli. Of the mechanisms involved, mitochondrial permeability transition appears to be associated mainly with necrosis, whereas the release of caspase activating proteins during early apoptosis is regulated primarily by the Bcl-2 family of proteins. However, there is increasing evidence for interaction and co-operation between these two mechanisms. The multiple mechanisms of mitochondrial permeabilization may explain diversities in the response of mitochondria to numerous apoptotic stimuli in different types of cells.     

Serglycin proteoglycan promotes apoptotic versus necrotic cell death in mast cells

The mechanisms that govern whether a cell dies by apoptosis or necrosis are not fully understood. Here we show that serglycin, a secretory granule proteoglycan of hematopoietic cells, can have a major impact on this decision. Wild type and serglycin(-/-) mast cells were equally sensitive to a range of cell death-inducing regimens. However, whereas wild type mast cells underwent apoptotic cell death, serglycin(-/-) cells died predominantly by necrosis. Investigations of the underlying mechanism revealed that cell death was accompanied by leakage of secretory granule compounds into the cytosol and that the necrotic phenotype of serglycin(-/-) mast cells was linked to defective degradation of poly(ADP-ribose) polymerase-1. Cells lacking mouse mast cell protease 6, a major serglycin-associated protease, exhibited similar defects in apoptosis as observed in serglycin(-/-) cells, indicating that the pro-apoptotic function of serglycin is due to downstream effects of proteases that are complex-bound to serglycin. Together, these findings implicate serglycin in promoting apoptotic versus necrotic cell death.     

Thapsigargin increases apoptotic cell death in human hepatoma BEL—7404 cells

Effects of thapsigargin,an inhibitor of Ca^2 -ATPase in surface of endoplasmic reticulum,on apoptotic cell death were studied in human hepatoma cells of BEL-7404 cell line by using both flow cytometry and electron microscopy.Propidium iodide staining and flow cytometry revealed that in the serum-free condition,thapsigargin increased the rate of apoptosis of BEL-7404 cells in a dose-dependent manner.Prolongation of the period of serum-free condition enhanced the apoptosis induced by thapsigargin treatment.Morphological observation with electron microscope further demonstrated that chromatin condensation and fragmentation,apoptotic bodies existed in TG-treated cells,supporting that thapsigargin is a potent activator of apoptosis in the cells.     

Clusterin protects granulosa cells from apoptotic cell death during follicular atresia

Clusterin expression is associated with programmed cell death (apoptosis) in many cell types but its exact role has not yet been defined. This study was carried out to determine the cellular localization of clusterin in the ovary and its functional role in the apoptotic cell death of ovarian follicles. A homogenous population of healthy and atretic follicles was obtained by treating immature rats with pregnant mare serum gonadotropin (PMSG). Apoptotic cell death was evaluated by TUNEL. Clusterin expression in the healthy and atretic follicles was examined by immunohistochemical and Western blot analyses, and gene expression was examined by Northern blot analysis. Clusterin protein and its mRNA are only expressed in granulosa cells of atretic follicles obtained from PMSG-treated rats on day 5 of the treatment. Healthy follicles from PMSG-treated rats on day 2 of the treatment do not express clusterin. Theca and stroma cells of both healthy and atretic follicles showed no signs of apoptosis and did not express clusterin. Withdrawal of trophic support from granulosa cells in cultures to induce apoptosis resulted in a dramatic increase in the levels of clusterin and its mRNA compared to cells cultured in serum-supplemented medium. In an attempt to establish the functional role of clusterin in the apoptotic cell death of ovarian follicles, the biosynthesis of clusterin in granulosa cells of healthy follicles was blocked by treatment of cells with antisense oligonucleotide to its cDNA. Treatment of granulosa cells with the antisense oligonucleotide resulted in an increase in the apoptotic cell death compared to the control. These findings indicate that depletion of clusterin can lead to the programmed cell death in ovary, suggesting a functional role for this protein in follicular atresia.     

Exposure to the anti-TNF-alpha drug thalidomide induces apoptotic cell death in human T leukemic cells.

TNF-alpha is a pro-inflammatory cytokine that plays a key role in disorders due to HIV-1 infection and replication such as Kaposi sarcoma, wasting, aphthous ulcerations and progression to AIDS. The controversial drug thalidomide is anti-inflammatory, anti-angiogenic and a selective inhibitor of TNF-alpha that is being studied as a treatment for HIV-1-related disorders, immune disorders and cancer. The cellular and molecular mechanism of thalidomide is unclear despite renewed clinical interest in the drug. Previous data from this laboratory indicate that thalidomide decreases cell growth and cell-cell interactions of human T leukemic cells. The specific aim of the present study is to determine whether thalidomide administration induces cell death via apoptosis. Low dose thalidomide treatment of human T leukemic cells exhibited rapid increases in caspase-3 activity, annexin V-FITC binding and DNA disintegration that is characteristic of apoptosis. These data indicate that low doses of thalidomide signal human T leukemic cells to die by apoptosis, which is a possible method of altering inflammatory cells and inflammatory activities.     

Effects of polysaccharides derived from Orostachys japonicus on induction of cell cycle arrest and apoptotic cell death in human colon cancer cells

Crude Orostachys japonicus polysaccharide extract (OJP) was prepared by hot steam extraction. Polysaccharides (OJPI) were separated from OJP by gel filtration chromatography and phenol-sulfuric acid assay. The average molecular weight of the OJPI was 30-50 kDa. The anti-proliferative effect of OJPI on HT-29 human colon cancer cells was investigated via morphology study, cell viability assay, apoptosis assay, cell cycle analysis, and cDNA microarray. OJPI inhibited proliferation and growth of HT29 cells and also stimulated apoptosis in a dose- and time-dependent manner. In cell cycle analysis, treatment with OJPI resulted in a marked increase of cells in the G0 (sub G1) and G2/M phases. To screen for genes involved in the induction of cell cycle arrest and apoptosis, the gene expression profiles of HT-29 cells treated with OJPI were examined by cDNA microarray, revealing that a number of genes were up- or down-regulated by OJPI. Whereas several genes involved in anti-apoptosis, cell proliferation and growth, and cell cycle regulation were down-regulated, expression levels of several genes involved in apoptosis, tumor suppression, and other signal transduction events were up-regulated. These results suggest that OJPI inhibits the growth of HT-29 human colon cancer cells by various apoptosis-aiding activities as well as apoptosis itself. Therefore, OJPI deserve further development as an effective agent exhibiting anticancer activity.     

Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases

 A new system to study programmed cell death in plants is described. Tomato (Lycopersicon esculentum Mill.) suspension cells were induced to undergo programmed cell death by treatment with known inducers of apoptosis in mammalian cells. This chemical-induced cell death was accompanied by the characteristic features of apoptosis in animal cells, such as typical changes in nuclear morphology, the fragmentation of the nucleus and DNA fragmentation. In search of processes involved in plant apoptotic cell death, specific enzyme inhibitors were tested for cell-death-inhibiting activity. Our results showed that proteolysis plays a crucial role in apoptosis in plants. Furthermore, caspase-specific peptide inhibitors were found to be potent inhibitors of the chemical-induced cell death in tomato cells, indicating that, as in animal systems, caspase-like proteases are involved in the apoptotic cell death pathway in plants. Received: 5 August 1999 / Accepted: 14 March 2000     

Conformational diseases and ER stress-mediated cell death: apoptotic cell death and autophagic cell death

The expanded polyglutamine (polyQ) tracts observed in autosomal dominant neurodegenerative disorders have the tendency to form intracellular aggregates, thus enhancing apoptotic cell death and the formation of autophagic vesicles. PolyQ accumulation inhibits the ER-associated degradation system (ERAD) resulting in reduced retrotranslocation from the ER and increased accumulation of misfolded proteins in the lumen of ER. Autophagy is an early cellular defense mechanism associated with ER stress, but prolonged ER stress may induce autophagic cell death, with destruction of cellular components and apoptotic cell death. Endoplasmic reticulum (ER) stress may be the key signal for both of these events.     

Induction of apoptotic cell death by putrescine

The polyamines are essential for cellular growth and differentiation. Ornithine decarboxylase (ODC), which catalyses the first step in the biosynthesis of the polyamines, has a very fast turnover and is subject to a strong feedback control by the polyamines. In the present study, we show that overexpression of a metabolically stable ODC in CHO cells induced a massive cell death unless the cells were grown in the presence of the ODC inhibitor alpha-difluoromethylornithine (DFMO). Cells overexpressing wild-type (unstable) ODC, on the other hand, were not dependent on the presence of DFMO for their growth. The induction of cell death was correlated with a dramatic increase in cellular putrescine levels. Analysis using flow cytometry revealed perturbed cell cycle kinetics, with a large accumulation of cells with sub-G1 amounts of DNA, which is a typical sign of apoptosis. Another strong indication of apoptosis was the finding that one of the key enzymes in the apoptotic process, caspase-3, was induced when DFMO was omitted from the growth medium. Furthermore, inhibition of the caspase activity significantly reduced the recruitment of cells to the sub-G1 fraction. In conclusion, deregulation of polyamine homeostasis may negatively affect cell proliferation and eventually lead to cell death by apoptosis if putrescine levels become too high.     

Intracellular bacteriolysis triggers a massive apoptotic cell death in Shigella-infected epithelial cells

Infected epithelial cells, which act as a first barrier against pathogens, seldom undergo apoptosis. Rather, infected epithelial cells undergo a slow cell death that displays hallmarks of necrosis. Here, we demonstrate that rapid intracellular lysis of Shigella flexneri, provoked by either the use of a diaminopimelic acid auxotroph mutant or treatment of infected cells with antibiotics of the beta-lactam family, resulted in a massive and rapid induction of apoptotic cell death. This intracellular bacteriolysis-mediated apoptotic death (IBAD) was characterized by the specific involvement of the mitochondrial-dependent cytochrome c/Apaf-1 axis that resulted in the activation of caspases-3, -6 and -9. Importantly, Bcl-2 family members and the NF-kappaB pathway seemed to be critical modulators of IBAD. Finally, we identified that IBAD was also triggered by Salmonella enterica serovar Typhimurium but not by the Gram-positive bacteria, Listeria monocytogenes. Together, our results demonstrate that, contrary to previous findings, epithelial cells are intrinsically able to mount an efficient apoptotic cell death response following infection. Indeed, apoptosis in normal circumstances is masked by powerful anti-apoptotic mechanisms, which are overcome in IBAD. Our results also uncover an unexpected consequence of the treatment of infected cells with certain classes of antibiotics.     

Preferential Fas-mediated apoptotic execution at G1 phase: the resistance of mitotic cells to the cell death

Apoptosis is induced by various stresses generated from the extracellular and intracellular environments. The fidelity of the cell cycle is monitored by surveillance mechanisms that arrest its further progression if any crucial process has not been completed or damages are sustained, and then the cells with problems undergo apoptosis. Although the molecular mechanisms involved in the regulation of the cell cycle and that of apoptosis have been elucidated, the links between them are not clear, especially that between cell cycle and death receptor-mediated apoptosis. By using the HeLa.S-Fucci (fluorescent ubiquitination-based cell cycle indicator) cells, we investigated the relationship between the cell cycle progression and apoptotic execution. To monitor apoptotic execution during cell cycle progression, we observed the cells after induction of apoptosis with time-lapse fluorescent microscopy. About 70% of Fas-mediated apoptotic cells were present at G₁ phase and about 20% of cells died immediately after cytokinesis, whereas more than 60% of etoposide-induced apoptotic cells were at S/G₂ phases in random culture of the cells. These results were confirmed by using synchronized culture of the cells. Furthermore, mitotic cells showed the resistance to Fas-mediated apoptosis. In conclusion, these findings suggest that apoptotic execution is dependent on cell cycle phase and Fas-mediated apoptosis preferentially occurs at G₁ phase.     

Apomorphine attenuates 6-hydroxydopamine-induced apoptotic cell death in SH-SY5Y cells

Abstract

Enhanced oxidative stress is implicated in the pathogenesis of Parkinson's disease. The catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) induces the production of reactive oxygen species (ROS), leading to neuronal cell death. On the other hand, apomorphine, a dopamine D1/D2 receptor agonist and known as a potent antioxidant, has been reported to have a neuroprotective effect. In the present study, we investigated the effect of apomorphine on 6-OHDA-induced apoptotic cell death using the human dopaminergic neuroblastoma cell line, SH-SY5Y. The co-treatment of cells with apomorphine significantly attenuated 6-OHDA-induced ROS generation, the phosphorylation of c-Jun N-terminal kinase (JNK), DNA fragmentation and subsequent apoptotic cell death. In addition, pretreatment with apomorphine for 24 h and the following concomitant treatment enhanced the protective effects against 6-OHDA-induced toxicity except for the attenuation of JNK phosphorylation. We also demonstrated that pretreatment alone with apomorphine for 24 h prior to the exposure confers resistance against 6-OHDA-induced cell toxicity. These findings suggested that apomorphine acts principally as a radical scavenger to suppress the level of ROS and ROS-stimulated apoptotic signaling pathway, whereas the other mechanisms might be involved in the protective effects.